GFAT is an important enzyme catalyzing the conversion of fructose-6-phosphate into glucosamine-6-phosphate, which in a living organism is the rate-determining step in the hexosamine biosynthesis pathway. Recently, McKnight et al. have disclosed a human GFAT gene containing 681 amino acid residues (McKnight, G. L., et al., J. Biol. Chem., 267, 25208–25212 (1992)). Thereafter, Nishi et al. disclosed a novel human GFAT gene (U.S. Pat. No. 5,876,713).
Inhibitors of GFAT activity are thought to promote uptake of glucose into cells and to lower blood glucose level. Therefore, the inhibitors are expected to find use as drugs for treating diabetes. Uptake of glucose into cells is promoted by insulin, and the glucose in the cells is metabolized by the glycolytic pathway, whereby ATP is accumulated as an energy source. However, when uptake of glucose is excessive, fructose-6-phosphate, which is a glucose metabolite, enters the hexosamine biosynthesis pathway. Fructose-6-phosphate in the hexosamine biosynthetic pathway is converted into glucosamine-6-phosphate by the action of GFAT.
Metabolites of glucosamine-6-phosphate have been reported to prevent glucose transporters from transferring to cell membranes, resulting in suppression of uptake of glucose into cells (FASEB J., 5, 3031–3036 (1991); Diabetologia, 38, 518–524 (1995); J. Biol. Chem., 266, 10155–10161 (1991); J. Biol. Chem., 266, 4706–4712 (1991); and Endocrinology, 136, 2809–2816 (1995)).
Within the glucose metabolism pathway, the hexosamine biosynthetic pathway is considered to play a role as a feedback mechanism with respect to excessive uptake of glucose. In the hexosamine biosynthetic pathway, GFAT is important as a rate-determining enzyme. GFAT activity is known to be generally high in patients suffering non-insulin-dependent (type 2) diabetes mellitus, and GFAT activity is reported to be one of the causes of high blood glucose levels (Diabetes, 45, 302–307 (1996)).
In addition to human GFAT (J. Biol. Chem., 267, 25208–25212 (1992): U.S. Pat. No. 5,876,713), mouse GFAT, yeast GFAT, and Escherichia coli GFAT have been reported. These GFATs are highly homologous to human GFAT. However, at present, the presence of novel GFAT that is specific to skeletal muscle, which is an important tissue for glucose metabolism, is not known.
Isolation of a novel GFAT gene exhibiting GFAT activity enables elucidation of the regulatory function of GFAT in the hexosamine biosynthetic pathway. Isolation of a GFAT gene that is specifically expressed in a tissue such as skeletal muscle enables further elucidation of the glucose metabolism mechanism in the tissue. In addition, discovery of specific candidate compounds exhibiting an inhibition activity against a GFAT protein, the protein being an expression product having an amino acid sequence encoded by such a GFAT gene, could lead to development of a hypoglycemic drug having a novel action mechanism and contributing to prevention and treatment of diabetes.
In view of the foregoing, an object of the present invention is to isolate such a novel GFAT gene, particularly a GFAT gene which is specifically expressed in skeletal muscle.